In recent years, non-antigenic water-soluble polymers, such as polyethylene glycol (“PEG”), have been used for the covalent modification of polypeptides of therapeutic and diagnostic importance. PEG is a polymer that is nontoxic, nonimmunogenic, highly water soluble, and readily cleared from the body. PEG has many applications and is commonly used in foods, cosmetics, beverages, and prescription medicines. Pharmaceutical grade PEGs are approved for use in the United States by the FDA and are widely used as biopharmaceutical carriers, given their high degree of biocompatibility. PEGylation can modify certain characteristics of biopharmaceuticals without altering their function, thereby enhancing the therapeutic effect.
Generally, polyethylene glycol molecules are connected to the protein via a reactive group found on the protein. Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such attachment. PEG can be coupled to active biopharmaceuticals through the hydroxyl groups at the ends of the chain using a variety of chemical methods. For example, covalent attachment of PEG to therapeutic polypeptides such as interleukins (Knauf, M. J. et al., J. Biol. Chem. 1988, 263, 15,064; Tsutsumi, Y. et al., J. Controlled Release 1995, 33, 447), interferons (Kita, Y. et al., Drug Des. Delivery 1990, 6, 157), catalase (Abuchowski, A. et al., J. Biol. Chem. 1977, 252, 3, 582), superoxide dismutase (Beauchamp, C. O. et al., Anal. Biochem. 1983, 131, 25), and adenosine deaminase (Chen, R. et al., Biochim. Biophy. Acta 1981, 660, 293), has been reported to extend their half life in vivo, and/or reduce their immunogenicity and antigenicity.
PEG molecules have been attached through amino groups on polypeptides using methoxylated PEG (“mPEG”) having different reactive moieties. Such polymers include mPEG-succinimidyl succinate, mPEG-succinimidyl carbonate, mPEG-imidate, and mPEG-cyanuric chloride. Alternatively, site-specific pegylation at the N-terminus, side chain and C-terminus of a potent analog of growth hormone-releasing factor has been performed through solid-phase synthesis (Felix, A. M. et al., Int. J. Peptide Protein Res. 1995, 46, 253). Site specific pegylation at the N-terminus has also been performed using aldehyde-activated PEG; however such reactions require long reaction times and are heavily dependent on pH. For example, the reaction requires from 18 to 36 hours and is generally specific only at acidic pH, becoming random at neural or higher pH (see e.g., U.S. Pat. Nos. 6,077,939 and 5,985,265, each of which is hereby incorporated by reference in its entirety). This limits the available peptides to those that can withstand prolonged acidic conditions.
An additional method used involved attaching a peptide to extremities of liposomal surface-grafted PEG chains in a site-specific manner through a reactive aldehyde group at the N-terminus generated by sodium periodate oxidation of N-terminal threonine (Zalipsky, S. et al., Bioconj. Chem. 1995, 6, 705). However, this method is limited to polypeptides with N-terminal serine or threonine residues.
Enzyme-assisted methods for introducing activated groups specifically at the C-terminus of a polypeptide have also been described (Schwarz, A. et al., Methods Enzymol. 1990, 184, 160; Rose, K. et al., Bioconjugate Chem. 1991, 2, 154; Gaertner, H. F. et al., J. Biol. Chem. 1994, 269, 7224). Typically, these active groups can be hydrazide, aldehyde, and aromatic-amino groups for subsequent attachment of functional probes to polypeptides.
Site-specific mutagenesis is a further approach which has been used to prepare polypeptides for site-specific polymer attachment. WO 90/12874 describes the site-directed pegylation of proteins modified by the insertion of cysteine residues or the substitution of other residues for cysteine residues. This publication also describes the preparation of mPEG-erythropoietin (“mPEG-EPO”) by reacting a cysteine-specific mPEG derivative with a recombinantly introduced cysteine residue on EPO. Similarly, interleukin-2 was pegylated at its glycosylation site after site-directed mutagenesis (Goodson, R. J. et al., Bio/Technology 1990, 8, 343).
Glycoproteins provide carbohydrates as additional target sites for modification. The enzyme peroxidase has been modified with PEG-diamine through its carbohydrate moiety (Urrutiogoity, M. et al., Biocatalysis 1989, 2, 145). WO 94/28024 describes the methods for preparing mPEG-EPO through periodate-oxidized carbohydrate. The chemistry involved was hydrazone formation by reacting mPEG-hydrazide with aldehyde groups of the carbohydrate moiety on EPO. This type of modification generates reactive aldehyde groups through an oxidation step, which potentially can oxidize various types of sugar residues in the carbohydrate moiety and some amino acid residues in the polypeptide, such as methionine.
Erythropoietin
An exemplary protein that demonstrates the need for improved pegylation methods is erythropoietin. Erythropoiesis is the production of red blood cells, which occurs to offset cell destruction. Erythropoiesis is a controlled physiological mechanism that enables sufficient red blood cells to be available for proper tissue oxygenation. Naturally occurring human erythropoietin (hEPO) is a polypeptide produced in the kidney and is the humoral plasma factor which stimulates red blood cell production (Carnot, P and Deflandre, C (1906) C.R. Acad. Sci. 143: 432; Erslev, A J (1953 Blood 8: 349; Reissmann, K R (1950) Blood 5: 372; Jacobson, L 0, Goldwasser, E, Freid, W and Plzak, L F (1957) Nature 179: 6331-4). Naturally occurring EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow and exerts its biological activity by binding to receptors on erythroid precursors (Krantz, B S (1991) Blood 77: 419).
Erythropoietin has been manufactured biosynthetically using recombinant DNA technology (Egrie, J C, Strickland, T W, Lane, J et al. (1986) Immunobiol. 72: 213-224) and is the product of a cloned human EPO gene inserted into and expressed in the ovarian tissue cells of the Chinese hamster (CHO cells). The primary structure of the predominant, fully processed form of hEPO is illustrated in SEQ ID NO:1. There are two disulfide bridges between Cys7-Cys161 and Cys29-Cys33. The molecular weight of the polypeptide chain of EPO without sugar moieties is 18,236 Da. In the intact EPO molecule (molecular weight of about 33 kD), approximately 40% of the molecular weight is accounted for by the carbohydrate groups that glycosylate the protein at glycosylation sites on the protein (Sasaki, H, Bothner, B, Dell, A and Fukuda, M (1987) J. Biol. Chem. 262: 12059).
Because human erythropoietin is essential in red blood cell formation, the hormone is useful in the treatment of blood disorders characterized by low or defective red blood cell production and other diseases for which expansion of red blood cell production would be beneficial to the patient. For example, EPO has been used in the treatment of anemia in chronic renal failure patients (CRF) (Eschbach, J W, Egri, J C, Downing, M R et al. (1987) NEJM 316: 73-78; Eschbach, J W, Abdulhadi, M H, Browne, J K et al. (1989) Ann. Intern. Med. 111: 992; Egrie, J C, Eschbach, J W, McGuire, T, Adamson, J W (1988) Kidney Intl. 33: 262; Lim, V S, Degowin, R L, Zavala, D et al. (1989) Ann. Intern. Med. 110: 108-114) and in AIDS and cancer patients undergoing chemotherapy (Danna, R P, Rudnick, S A, Abels, R I In: M B, Garnick, ed. Erythropoietin in Clinical Applications—An International Perspective. New York, N.Y.: Marcel Dekker; 1990: p. 301-324).
However, the bioavailability of commercially available protein therapeutics such as EPO is limited by their short plasma half-life and susceptibility to protease degradation. These shortcomings prevent them from attaining maximum clinical potency and generally require more frequent treatments or administration of greater amounts of drug which can result in increased frequency and severity of side effects and lack of patient compliance with the treatment schedule. Proteins, including native EPO and its derivatized and modified forms, are also generally formulated with albumin (HSA or serum), and stored and transported at reduced temperature to help maintain product stability through use. HSA serum-containing formulations are undesirable because of the risk of contamination by human infectious agents and the high costs associated with pharmaceutical grade HSA and related bioassays.
ARANESP® (darbepoietin alfa) is a commercially available EPO derivative. It is a 165-amino acid protein that differs from recombinant human erythropoietin in containing 5 N-linked oligosaccharide chains, whereas recombinant human erythropoietin contains 3 chains. The two additional N-glycosylation sites result from amino acid substitutions in the erythropoietin peptide backbone. The additional carbohydrate chains increase the approximate molecular weight of the glycoprotein from 30,000 to 37,000 daltons. ARANESP® is supplied in two formulations with different excipients, one containing polysorbate 80 and another containing albumin (HSA), a derivative of human blood.
Pegylated proteins, e.g., EPO derivatives, have been disclosed (U.S. Publication Nos. 2002/0115833, 2003/0120045 and 2003/0166566) However, the processes used to make these compositions are difficult to implement and control, costly, use toxic compounds in the synthesis, or have other quality control problems. Moreover, they are not known to generate polypeptide conjugates that have biological activity greater than natural function of the unmodified polypeptide.
As such, there remains an acute need for a pegylated composition, and/or method of production thereof, that is easier and less costly to implement and, most importantly, easier to control in order to produce a predictable and consistent product.
With particular respect to EPO, formulations are required that have improved plasma half-lives, increased activity, and decreased susceptibility to protease degradation relative to currently available formulations. Moreover, such formulations should optimally be able to be packaged, shipped, and stored in protein-free formulations and/or under standard conditions.
All scientific publications including patent documents cited herein are incorporated by reference in their entirety for all purposes.